| The purple sea urchin (Strongylocentrotus purpuratus) harbors a set of highly diverse immune genes (∼50-60 per genome), called Sp185/333, and their encoded proteins possess an additional level of diversity (∼200-260 per genome). The specific function(s) of the Sp185/333 proteins are still unknown. However, the proteins are expressed in two sub-types of immune cells, called coelomocytes. Specifically, the proteins are expressed in perinuclear vesicles of type 2 polygonal phagocytes and small phagocytes, as well as on the cell surface of small phagocytes. Following in vivo immune challenge, the proportion of both Sp185/333-positive (Sp185/333+) small and polygonal cells residing in the central cavity significantly increases, compared to the proportion of these cells prior to challenge.;Aggregated phagocytes have been shown to include Sp185/333+ cells. However, it is not known whether Sp185/333 gene expression, Sp185/333+ cells and Sp185/333 protein expression are found in various organs and tissues throughout the body in addition to expression in coelomocytes. Therefore, we examined the Sp185/333 transcript level as well as expression of Sp185/333 proteins associated with cells in the pharynx, esophagus, intestine, gonad and axial organ, in addition to examining coelomocytes from immunoquiescent (immune down-regulated) and immune challenged sea urchins.;Results indicate that the level of Sp185/333 transcripts increase in coelomocytes, intestine and axial organ after immune challenge compared to the same organ samples in immunoquiescent (immune-downregulated) animals. Sp185/333+ cells are observed in all organs. The proportion of Sp185/333+ cells increases in axial organ and coelomocytes in immune challenged animals compared to the same organ samples in non-challenged animals. The concentration of Sp185/333 proteins in organs increases in intestine, pharynx, esophagus and axial organ as well as coelomocytes collected from the coelomic cavity in immune challenged animals compared to the non-challenged animals. The major Sp185/333 proteins in organs range from 250 kDa, which is a wider range than what is typically observed in the coelomocytes (∼90 to < 250 kDa). There are no observable changes in Sp185/333 repertoires in organs from immune challenged animals vs. immunoquiescent animals.;To measure if coelomocytes are capable of producing an increase in the proportion of Sp185/333+ cells in culture, a short-term method was devised for culturing coelomocytes collected from the coelomic cavity of purple sea urchins. Viability results for coelomocytes in culture from 1–6 days ranges from 57.0 to 91.6 %. Syncytia-like structures form in cultured coelomocytes beginning at 3 h and up to 24 h. Results indicate that aggregate formation occurs at a faster rate in response to lipopolysaccharide (LPS). All of the phagocytes are incorporated into syncytia-like structures in the presence of LPS by 3 h, however most of these cells at this time do not express the Sp185/333 proteins. At 24 h however, almost all of the nuclei in the syncytia-like structures are localized with the Sp185/333 proteins.;RNA interference (RNAi) was implemented on cultured coelomocytes and in animals, in vivo and in vivo, to understand more about the function(s) of the genes. Results from double stranded (ds) Sp185/333 RNA (ds185/333)-treated phagocytes in vitro indicate that the level of Sp185/333 transcripts is knocked down in coelomocytes from 1 to 3 d. Results from in vivo RNAi analysis indicates that the Sp185/333 gene expression is knocked down in phagocytes beginning at 13 h after ds1853/333 injection. Overall, these results indicate that in vitro and in vivo RNAi is feasible sea urchins, and further research can be implemented through this approach to understand the functions of this immune gene family. |
